Department of Chemistry, Indiana University, Bloomington, Indiana, United States of America.
PLoS One. 2010 Aug 24;5(8):e12368. doi: 10.1371/journal.pone.0012368.
In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 5' or 2' OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated.
METHODOLOGY/PRINCIPAL FINDINGS: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5' phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxy)ribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A(2)>A(1)>A(3)>A(4)>A(0)>A(6)>A(8)>A(10) and T(2)>T(3)>T(4)>T(6) approximately T(1)>T(8)>T(10). Bridging T's generally gave higher yield of ligated product than bridging A's. ATP concentrations above 33 microM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (>90%) joined a correctly paired, or TratioG wobble-paired, substrate on the 3' side of the ligation junction while discriminating approximately 100-fold against most mispaired substrates. Tethered dC and dG gave the highest ligation rates and yields, followed by tethered deoxyinosine (dI) and dT, with the slowest reactions for tethered dA. The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates. The "universal" analog 5-nitroindole (dNI) did not support ligation when used as the tethered nucleotide.
CONCLUSIONS/SIGNIFICANCE: Our results reveal a novel activity for T4 DNA ligase (template-directed ligation of a tethered mononucleotide) and establish this partition scheme as being suitable for the selection of ribozymes that phosphorylate mononucleoside substrates.
体外选择小分子代谢物（如游离核苷）的激酶核酶，将需要区分活性和非活性 RNA 种类的分配系统。虽然磷酸转移的核酸催化已被证实可用于寡核苷酸底物的 5'或 2' OH 的磷酸化，但可扩散小分子的磷酸化尚未得到证明。
方法/主要发现：本研究证明了 T4 DNA 连接酶能够捕获与连接的单脱氧核苷获得 5' 磷酸的 RNA 链。因此，该连接反应模拟了核苷激酶（脱氧）核酶选择的分配步骤。与带缺口的完全双链 DNA 相比，带有连接物的底物的连接反应速度要慢得多，尽管连接物中的脱氧核苷酸在连接物中与沃森-克里克碱基配对。当桥接模板链在柔性连接物对面包含未配对的间隔核苷酸时，连接显著增加，根据趋势：A(2)>A(1)>A(3)>A(4)>A(0)>A(6)>A(8)>A(10)和 T(2)>T(3)>T(4)>T(6)大致 T(1)>T(8)>T(10)。桥接 T 通常比桥接 A 产生更高产量的连接产物。33μM 以上的 ATP 浓度会积累腺苷酸化中间产物并降低缺口封闭产物的产量，这可能是由于分离的酶重新腺苷酸化所致。在优化条件下，T4 DNA 连接酶有效地（>90%）连接在连接点 3' 侧的正确配对或 TratioG 摆动配对的底物，而对大多数错配底物的选择性约为 100 倍。带连接物的 dC 和 dG 具有最高的连接率和产量，其次是带连接物的脱氧肌苷（dI）和 dT，带连接物的 dA 反应最慢。在具有多种底物的复杂反应混合物中，通过连接酶介导的捕获观察到相同的动力学趋势。作为连接物核苷酸使用的通用类似物 5-硝基吲哚（dNI）不支持连接。
结论/意义：我们的结果揭示了 T4 DNA 连接酶的一种新活性（模板指导的连接物单核苷酸），并建立了这种分配方案，适合选择磷酸化单核苷酸底物的核酶。
